Netrin receptors

ABSTRACT

The invention provides methods and compositions relating to vertebrate UNC-5 proteins which function as receptor proteins for netrins, a family of cell guidance proteins. The proteins may be produced recombinantly from transformed host cells from the disclosed vertebrate UNC-5 encoding nucleic acid or purified from human cells. The invention provides specific hybridization probes and primers capable of specifically hybridizing with the disclosed vertebrate unc-5 gene, vertebrate UNC-5-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis, therapy and in the biopharmaceutical industry.

[0001] The research carried out in the subject application was supportedin part by grants from the National Institutes of Health. The governmentmay have rights in any patent issuing on this application.

INTRODUCTION

[0002] 1. Field of the Invention

[0003] The field of this invention is proteins which regulate vertebratecell guidance.

[0004] 2. Background

[0005] In the developing nervous system, migrating cells and axons areguided to their targets by cues in the extracellular environment. Thenetrins are a family of phylogenetically-conserved guidance cues thatcan function as diffusible attractants and repellents for differentclasses of cells and axons¹⁻¹⁰. Recent studies in vertebrates, insectsand nematodes have implicated members of the DCC subfamily of theimmunoglobulin (Ig) superfamily as receptors involved in migrationstoward netrin soures^(6.11-13). The mechanisms that direct migrationsaway from netrin sources (presumed repulsions) are less well understood.In Caenorhabditis elegans, loss of unc-5 (which encodes thetransmembrane protein UNC-5¹⁴) function causes defects in thesemigrations^(15, 16), and ectopic expression of unc-5 in some neurons canredirect their axons away from a netrin source¹⁷. However, therelationship between UNC-5 and the netrins has not been defined. Wedisclose herein vertebrate homologues of the C. elegans UNC-5, whichdefine a novel subfamily of the Ig superfamily, and whose mRNAs showprominent expression in various classes of differentiating neurons andwe disclose that these vertebrate UNC-5 homologues are vertebratenetrin-binding proteins.

SUMMARY OF THE INVENTION

[0006] The invention provides methods and compositions relating tovertebrate UNC-5 proteins, related nucleic acids, and protein domainsthereof having vertebrate UNC-5-specific activity. The proteins may beproduced recombinantly from transfected host cells from the subjectvertebrate UNC-5 encoding nucleic acids or purified from vertebratecells. The invention provides isolated vertebrate unc-5 hybridizationprobes and primers capable of specifically hybridizing with thedisclosed vertebrate unc-5 genes, vertebrate UNC-5-specific bindingagents such as specific antibodies, and methods of making and using thesubject compositions in diagnosis (e.g. genetic hybridization screensfor vertebrate unc-5 transcripts), therapy (e.g. gene therapy tomodulate vertebrate unc-5 gene expression) and in the biopharmaceuticalindustry (e.g. as immunogens, reagents for modulating cell guidance,reagents for screening chemical libraries for lead pharmacologicalagents, etc.).

DETAILED DESCRIPTION OF THE INVENTION

[0007] The nucleotide sequences of natural unc5h-1 cDNAs from rat andhuman are shown as SEQ ID NOS:1 and 2, respectively; and the conceptualtranslates are shown as SEQ ID NOS:5 and 6, respectively. The nucleotidesequences of natural unc5h-2 cDNAs from rat and human are shown as SEQID NOS:3 and 4, respectively; and the conceptual translates are shown asSEQ ID NOS:7 and 8, respectively. The vertebrate UNC-5 proteins of theinvention include incomplete translates of SEQ ID NOS:1, 2, 3 and 4 anddeletion mutants of SEQ ID NOS:5, 6, 7 and 8, which translates anddeletion mutants have vertebrate UNC-5-specific amino acid sequence andassay-discernable vertebrate UNC-5-specific binding specificity orfunction. Such active vertebrate UNC-5 deletion mutants, vertebrateUNC-5 peptides or protein domains comprise at least about 8, preferablyat least about 12, more preferably at least about 24 consecutiveresidues of SEQ ID NO:5, 6, 7 or 8. For examples, vertebrate UNC-5protein domains identified below are shown to provide protein-bindingdomains which are identified in and find use, inter alia, in solid-phasebinding assays as described below.

[0008] Vertebrate UNC-5-specific activity or function may be determinedby convenient in vitro, cell-based, or in vivo assays: e.g. in vitrobinding assays, cell culture assays, in animals (e.g. gene therapy,transgenics, etc.), etc. Binding assays encompass any assay where themolecular interaction of a vertebrate UNC-5 protein with a bindingtarget is evaluated. The binding target may be a natural extracellularbinding target such as a netrin protein, or other regulator thatdirectly modulates vertebrate UNC-5 activity or its localization; ornon-natural binding target such a specific immune protein such as anantibody, or an vertebrate UNC-5 specific agent such as those identifiedin screening assays such as described below. Vertebrate UNC-5-bindingspecificity may assayed by binding equilibrium constants (usually atleast about 10⁷M⁻¹, preferably at least about 10⁸M⁻¹, more preferably atleast about 10⁹M⁻¹), by the ability of the subject protein to functionas negative mutants in vertebrate UNC-5-expressing cells, to elicitvertebrate UNC-5 specific antibody in a heterologous mammalian host (e.ga rodent or rabbit), etc. In any event, the vertebrate UNC-5 bindingspecificity of the subject vertebrate UNC-5 proteins necessarilydistinguishes C. elegans UNC-5.

[0009] The claimed vertebrate UNC-5 proteins are isolated or pure: an“isolated” protein is unaccompanied by at least some of the materialwith which it is associated in its natural state, preferablyconstituting at least about 0.5%, and more preferably at least about 5%by weight of the total protein in a given sample and a pure proteinconstitutes at least about 90%, and preferably at least about 99% byweight of the total protein in a given sample. The vertebrate UNC-5proteins and protein domains may be synthesized, produced by recombinanttechnology, or purified from mammalian, preferably human cells. A widevariety of molecular and biochemical methods are available forbiochemical synthesis, molecular expression and purification of thesubject compositions, see e.g. Molecular Cloning, A Laboratory Manual(Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols inMolecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc.,Wiley-Interscience, NY) or that are otherwise known in the art.

[0010] The invention provides natural and non-natural vertebrateUNC-5-specific binding agents, methods of identifying and making suchagents, and their use in diagnosis, therapy and pharmaceuticaldevelopment. For example, vertebrate UNC-5-specific agents are useful ina variety of diagnostic and therapeutic applications. VertebrateUNC-5-specific binding agents include vertebrate UNC-5-specific ligands.such as netrins, and somatically recombined protein receptors likespecific antibodies or T-cell antigen receptors (see, e.g Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory) and other natural binding agents identified with assays suchas one-. two- and three-hybrid screens, non-natural binding agentsidentified in screens of chemical libraries such as described below,etc. For diagnostic uses, the binding agents are frequently labeled,such as with fluorescent, radioactive, chemiluminescent, or other easilydetectable molecules, either conjugated directly to the binding agent orconjugated to a probe specific for the binding agent. Agents ofparticular interest modulate vertebrate UNC-5 function, e.g. vertebrateUNC-5-dependent cell guidance; for example, isolated cells, wholetissues, or individuals may be treated with a vertebrate UNC-5 bindingagent to activate, inhibit, or alter vertebrate UNC-5-dependent cellguidance or function.

[0011] The invention provides UNC-5 related nucleic acids, which find awide variety of applications including use as translatable transcripts,hybridization probes, PCR primers, diagnostic nucleic acids, etc.; usein detecting the presence of unc-5 genes and gene transcripts and indetecting or amplifying nucleic acids encoding additional unc-5 homologsand UNC-5 structural analogs. The subject nucleic acids are ofsynthetic/non-natural sequences and/or are isolated, i.e. unaccompaniedby at least some of the material with which it is associated in itsnatural state, preferably constituting at least about 0.5%, preferablyat least about 5% by weight of total nucleic acid present in a givenfraction, and usually recombinant, meaning they comprise a non-naturalsequence or a natural sequence joined to nucleotide(s) other than thatwhich it is joined to on a natural chromosome. Nucleic acids comprisingthe nucleotide sequence of SEQ ID NO:1, 2, 3 or 4 or fragments thereof,contain such sequence or fragment at a terminus, immediately flanked bya sequence other than that which it is joined to on a naturalchromosome, or flanked by a native flanking region fewer than 10 kb,preferably fewer than 2 kb, which is at a terminus or is immediatelyflanked by a sequence other than that which it is joined to on a naturalchromosome. While the nucleic acids are usually RNA or DNA, it is oftenadvantageous to use nucleic acids comprising other bases or nucleotideanalogs to provide modified stability, etc.

[0012] The amino acid sequences of the disclosed vertebrate UNC-5proteins are used to back-translate vertebrate UNC-5 protein-encodingnucleic acids optimized for selected expression systems (Holler et al.(1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) orused to generate degenerate oligonucleotide primers and probes for usein the isolation of natural vertebrate UNC-5-encoding nucleic acidsequences (“GCG” software, Genetics Computer Group, Inc, Madison Wis).vertebrate UNC-5-encoding nucleic acids used in vertebrateUNC-5-expression vectors and incorporated into recombinant host cells,e.g. for expression and screening, transgenic animals, e.g. forfunctional studies such as the efficacy of candidate drugs for diseaseassociated with vertebrate UNC-5-modulated transcription, etc.

[0013] The invention also provides nucleic acid hybridization probes andreplication/amplification primers having a vertebrate UNC-5 cDNAspecific sequence contained in SEQ ID NO:1, 2, 3 or 4 and sufficient toeffect specific hybridization thereto (i.e. specifically hybridize withthe corresponding SEQ ID NO:1, 2, 3 or 4 in the presence of C. elegansunc-5 cDNA). Such primers or probes are at least 12, preferably at least24, more preferably at least 36 and most preferably at least 96 bases inlength. Demonstrating specific hybridization generally requiresstringent conditions, for example, hybridizing in a buffer comprising30% formamide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO₄, pH7.7, 0.001 M EDTA)buffer at a temperature of 42° C. and remaining bound when subject towashing at 42° C. with 0.2×SSPE; preferably hybridizing in a buffercomprising 50% formamide in 5×SSPE buffer at a temperature of 42° C. andremaining bound when subject to washing at 42° C. with 0.2×SSPE bufferat 42° C. vertebrate UNC-5 cDNA homologs can also be distinguished fromother protein using alignment algorithms, such as BLASTX (Altschul etal. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410).

[0014] Vertebrate unc-5 hybridization probes find use in identifyingwild-type and mutant vertebrate unc-5 alleles in clinical and laboratorysamples. Mutant alleles are used to generate allele-specificoligonucleotide (ASO) probes for high-throughput clinical diagnoses.Therapeutic vertebrate UNC-5 nucleic acids are used to modulate cellularexpression or intracellular concentration or availability of activevertebrate UNC-5. For example, vertebrate UNC-5 nucleic acids are alsoused to modulate cellular expression or intracellular concentration oravailability of active vertebrate UNC-5 protein. Vertebrate UNC-5inhibitory nucleic acids are typically antisense: single-strandedsequences comprising complements of the disclosed natural vertebrateUNC-5 coding sequences. Antisense modulation of the expression of agiven vertebrate UNC-5 protein may employ antisense nucleic acidsoperably linked to gene regulatory sequences. Cells are transfected witha vector comprising a vertebrate UNC-5 sequence with a promoter sequenceoriented such that transcription of the gene yields an antisensetranscript capable of binding to endogenous vertebrate UNC-5 encodingmRNA. Transcription of the antisense nucleic acid may be constitutive orinducible and the vector may provide for stable extrachromosomalmaintenance or integration. Alternatively, single-stranded antisensenucleic acids that bind to genomic DNA or MRNA encoding a givenvertebrate UNC-5 protein may be administered to the target cell, in ortemporarily isolated from a host, at a concentration that results in asubstantial reduction in expression of the targeted protein. Anenhancement in vertebrate UNC-5 expression is effected by introducinginto the targeted cell type vertebrate UNC-5 nucleic acids whichincrease the functional expression of the corresponding gene products.Such nucleic acids may be vertebrate UNC-5 expression vectors, vectorswhich upregulate the functional expression of an endogenous allele, orreplacement vectors for targeted correction of mutant alleles.Techniques for introducing the nucleic acids into viable cells are knownin the art and include retroviral-based transfection, viral coatprotein-liposome mediated transfection, etc.

[0015] The invention provides efficient methods of identifying agents,compounds or lead compounds for agents active at the level of avertebrate UNC-5 modulatable cellular function. Generally, thesescreening methods involve assaying for compounds which modulatevertebrate UNC-5 interaction with a natural vertebrate UNC-5 bindingtarget. A wide variety of assays for binding agents are providedincluding labeled in vitro protein-protein binding assays. immunoassays,cell based assays, animal based assay, etc. Preferred methods areamenable to automated, cost-effective high throughput screening ofchemical libraries for lead compounds. Such libraries encompasscandidate agents of numerous chemical classes, though typically they areorganic compounds; preferably small organic compounds and are obtainedfrom a wide variety of sources including libraries of synthetic ornatural compounds. Identified agents find use in the pharmaceuticalindustries for animal and human trials; for example, the agents may bederivatized and rescreened in in vitro and in vivo assays to optimizeactivity and minimize toxicity for pharmaceutical development.

[0016] In vitro binding assays employ a mixture of components includingvertebrate UNC-5 protein, which may be part of a fusion product withanother peptide or polypeptide, e.g. a tag for detection or anchoring,etc. The assay mixtures comprise a natural extracellular vertebrate UNC-binding target, such as a netrin. While native binding targets may beused, it is frequently preferred to use portions (e.g. peptides) thereofso long as the portion provides binding affinity and avidity to thesubject vertebrate UNC-5 protein conveniently measurable in the assay.The assay mixture also comprises a candidate pharmacological agent andtypically, a variety of other reagents such as salts, buffers, neutralproteins, e.g. albumin, detergents, protease inhibitors, nucleaseinhibitors, antimicrobial agents, etc. The mixture components can beadded in any order that provides for the requisite bindings andincubations may be performed at any temperature which facilitatesoptimal binding. The mixture is then incubated under conditions whereby,but for the presence of the candidate pharmacological agent, thevertebrate UNC-5 protein specifically binds the cellular binding target,portion or analog with a reference binding affinity. Incubation periodsare likewise selected for optimal binding but also minimized tofacilitate rapid, high-throughput screening.

[0017] After incubation, the agent-biased binding between the vertebrateUNC-5 protein and one or more binding targets is detected. A separationstep is often initially used to separate bound from unbound components.Separation may be effected by precipitation (e.g. TCA precipitation,immunoprecipitation, etc.), immobilization (e.g on a solid substrate),etc., followed by washing by, for examples, membrane filtration, gelchromatography (e.g. gel filtration, affinity, etc.). One of thecomponents usually comprises or is coupled to a label. The label mayprovide for direct detection such as radioactivity, luminescence,optical or electron density, etc. or indirect detection such as anepitope tag, an enzyme, etc. A variety of methods may be used to detectthe label depending on the nature of the label and other assaycomponents, e.g. through optical or electron density, radiativeemissions, nonradiative energy transfers, etc. or indirectly detectedwith antibody conjugates, etc. A difference in the binding affinity ofthe vertebrate UNC-5 protein to the target in the absence of the agentas compared with the binding affinity in the presence of the agentindicates that the agent modulates the binding of the vertebrate UNC-5protein to the vertebrate UNC-5 binding target. Analogously, in thecell-based transcription assay also described below, a difference in thevertebrate UNC-5 transcriptional induction in the presence and absenceof an agent indicates the agent modulates vertebrate UNC-5-inducedtranscription. A difference, as used herein, is statisticallysignificant and preferably represents at least a 50%, more preferably atleast a 90% difference.

[0018] The following experimental section and examples are offered byway of illustration and not by way of limitation.

EXPERIMENTAL

[0019] cDNAs encoding two rat homologues of UNC-5, termed UNC5H-1 (SEQID NO:1) and UNC5H-2 (SEQ ID NO:2), were isolated from an E18 rat braincDNA library (see Methods). The predicted proteins (SEQ ID NOS: 3 and 4)show sequence similarity with UNC-5 over their entire lengths, but aremore similar to one another (52% identity) than to UNC-5 (28% identityin each case). Like UNC-5¹⁴, both possess two predicted Ig-like domainsand two predicted thrombospondin type-1 repeats in their extracellulardomains, a predicted membrane spanning region, and a large intracellulardomain. The UNC5H proteins also each possess a signal sequence which,curiously, is lacking in UNC-5¹⁴. The predicted topology of the UNC5Hproteins in cell membranes was verified using recombinant versions ofthe proteins expressed in transfected cells and antibodies directedagainst the extracellular and intracellular domains (see Methods). Thecytoplasmic domains of the two UNC5H proteins do not contain obvioussignaling motifs, but do possess a small region of homology to ZonaOccludens-l (ZO-1), a protein that localizes to adherens junctions andis implicated in junction formation^(18, 19). ZO-1 containsPDZ-domains^(18, 19), structures implicated in protein clustering²⁰, butthe region of homology with UNC-5 homologues corresponds to a uniquesequence at the carboxy terminus of ZO-1. The homology between ZO-1 andC. elegans UNC-5 is less pronounced (and is not detected by computerBLAST search), but is nonetheless apparent when all four sequences arealigned.

[0020] To determine whether the UNC-5 homologues are candidates forreceptors involved in neuronal migration or axon guidance, we firstexamined the sites of expression of Unc5h-1 and Unc5h-2 by RNA in situhybridization in rat embryos. Unc5h-1 transcripts are detected at earlystages of neural tube development in the ventral spinal cord. Atembryonic day 11 (E11), when motoneurons are beginning to differentiatein that region²¹, transcripts are present throughout the ventral spinalcord, excluding the midline floor plate region, but are most intense inthe ventricular zone and at the lateral edges. At E12, prominentexpression is observed in the motor columns, but also extends moredorsally, and is now becoming excluded from the ventricular zone. Thismore dorsal expression appears transient, as expression by E13 isconfined to postmitotic cells in the ventral spinal cord, apparentlyincluding the motoneurons. Unc5h-2 transcripts are not detected atsignificant levels in the spinal cord until E14, when they are found inthe roof plate region. Unc5h-2 transcripts are, however, detected indeveloping sensory ganglia that flank the spinal cord, at low levels atE12, and at higher levels by E14. The expression of these two genes isthus observed in regions where differentiating neurons are undergoingaxonogenesis, consistent with a possible role in this process.

[0021] Expression of these genes is also observed at higher axial levelsof the nervous system, as well as in non-neural structures. At E13,Unc5h-1 is expressed in the basal plate (ventral neural tube) in thehindbrain and midbrain, in the developing hypothalamus and thalamus, andin the pallidum. Unc5h-2 expression at this stage is detected in thedorsal aspect of the developing optic cup, the nasal pits, apical ridgeof the limb bud, urogenital tubercle, and in restricted regions of themidbrain and caudal diencephalon. By E16, Unc5h-1 mRNA is also detectedat high levels in the entorhinal cortex and at lower levels throughoutthe cortex. Unc5h-2 is also detected at this stage at low levels in thecortex, and at high levels in hypertrophic chondrocytes. Expression ofthe two homologues persists postnatally, with, at postnatal day 10(P10), continued expression of both at low levels throughout the cortex,expression of both in distinct patterns in the septal area, and highlevel expression of Unc5h-1 in the developing hippocampus and entorhinalcortex. In addition, a prominent site of postnatal expression of bothgenes is in the cerebellum. Both are expressed in the inner granule celllayer, and Unc5h-2 is in addition expressed in the inner aspect of theexternal germinal layer, where granule cell precursors differentiateprior to migrating to their final destination in the inner granule celllayer^(22, 23). Thus, expression of Unc5h-2 in this region is associatedwith a prominent cell migration event in the developing cerebellum.

[0022] Although the expression patterns of the two UNC5H proteins weresuggestive of potential roles in cell or axon migration, to obtain moredirect evidence implicating them in mediating responses to netrins wetested whether netrin-1 can bind cells expressing these proteins.Transfected monkey kidney COS-1 cells or human embryonic kidney 293cells expressing either UNC5H-1 or UNC5H-2 showed significant binding ofnetrin-1 protein above background, as is also observed for transfectedcells expressing the netrin receptors DCC and neogenin, but not fortransfected cells expressing TAG-1or L1, two other members of the Igsuperfamily¹³. In these experiments, binding was performed in thepresence of soluble heparin, which eliminates non-specific binding ofnetrin-1 to the cells¹³ but does not evidently prevent binding to theUNC5 homologues. To verify, in the case of UNC5H-2, that exogenouslyadded heparin is not required for the interaction, we generated asoluble protein comprising the extracellular domain of UNC5H-2 fused tothe constant region (Fc) of a human immunogloblin molecule. ThisUNC5H-2-Fc fusion protein bound transfected 293 cells expressingnetrin-1 (some of which remains associated with the surface of thesecells^(3, 10)) in the absence of added heparin but did not show bindingto non-transfected cells, nor to cells expressing UNC5H-2 itself, DCC,or neogenin. The UNC5H-2-Fc fusion also did not bind transfected cellsexpressing F-spondin, an adhesive extracellular matrix protein made byfloor plate cells²⁴, or Semaphorin III, a chemorepellent for sensoryaxons at the stages that Unc5h-2 is expressed in sensory ganglia²⁵. Bothof these proteins, like netrin-1, are secreted but partition betweencell surfaces and the soluble fraction^(24,26). Thus, the interactionbetween netrin-1 and UNC5H-2 appears specific, and does not requireheparin nor reflect a generalized interaction with proteins thatassociate non-specifically with cell surfaces.

[0023] The affinity of UNC-5 homologues for netrin-1 was estimated inequilibrium binding experiments using netrin(VIoV)-Fc, a fusion of theamino terminal two-thirds of netrin-1 to the constant portion of humanIgG¹³. This netrin-1 derivative is bioactive but, unlike netrin-1, doesnot aggregate at high concentrations, and it binds DCC with a Kdcomparable to that of full length netrin-1¹³. Specific binding of netrin(VIoV)-Fc to each of the three UNC5 homologues showed saturation and thebinding curves were fitted to the Hill equation, yielding Kd values of19±0.8 nM and 3.4±1.0 nM for UNC5H1 and UNC5H2 respectively. Thesevalues are comparable to the Kd for the DCC-netrin (VIoV-Fc) interaction(˜5 nM), and are consistent with the effective dose for the axonoutgrowth promoting effects of netrin-1^(2,13).

[0024] Establishing the involvement of these vertebrate UNC5H proteinsin cell migration and axon guidance will require perturbing theirfunctions in vivo. In the meantime, however, our results are at leastconsistent with such an involvement, as these homologues are expressedby some populations of cells that are undergoing migrations or extendingaxons. For example, Unc5h1 is expressed by spinal motoneurons, whoseaxons are repelled in vitro by floor plate cells²⁷, and whose outgrowthin vitro can be suppressed by netrin-1. It is also expressed in theregion of trochlear motoneurons, which can be repelled by netrin-1⁴.Both Unc5h genes are also expressed in the developing cerebellum, whichis a site of extensive cell migration.

[0025] Although the in vivo functions of the UNC-5 homologues describedhere remain to be determined, our evidence that vertebrate UNC5Hproteins bind netrin-1 provides direct support for the idea that membersof this new subfamily of the Ig superfamily are netrin receptors. Thisidea was first proposed for C. elegans UNC-5, based on the findings thatunc-5 is required cell-autonomously for dorsal migrations that requirethe function of the netrin UNC-6¹⁴, and that ectopic expression of unc-5in neurons that normally project longitudinally or ventrally can steertheir axons dorsally¹⁷. Although consistent with the possibility thatUNC-5 is an UNC-6 receptor, these results are also consistent with arole for UNC-5 in modifying the function of a distinct UNC-6 receptor.The possibility of a modifier function was made more plausible byevidence that the DCC homologue UNC40, which is a putative UNC-6receptor involved in ventral migrations¹¹, is expressed by axons thatproject dorsally and is required for those projections^(11, 15, 16),suggesting that UNC-5 might function by switching an attractive netrinreceptor (UNC40) into a repulsive netrin receptor. However, our resultssuggest that UNC-5 also functions directly as a netrin receptor. A modelin which UNC-40 and UNC-5 can form a receptor complex but UNC-5 can alsofunction alone in transducing the UNC-6 netrin signal provides anexplanation for the observation that loss of unc-40 function results ina much less severe phenotype for dorsal migrations than do either lossof unc-5 or loss of unc-6 function^(15, 16).

[0026] Recent studies have demonstrated a remarkable phylogeneticconservation in function of netrin proteins in guiding axons towards asource of netrin at the midline of the nervous systems of nematodes,flies and vertebrates^(1,7,8,9), as well as a conserved role for membersof the DCC subfamily of the Ig superfamily in mediating the axonalresponses that underlie those guidance events^(11, 12, 13). Theidentification of vertebrate homologues of UNC-5, and the evidence thatthey are netrin-binding proteins, suggests that the signaling mechanismsthrough which netrins elicit repulsive responses are also conserved.

[0027] Isolation of rat UNC-5 homologues, and in situ hybridization. Asearch of the human expressed sequence tag (EST) databases revealed asmall sequence (Genbank accession number R11880) with distant similarityto the carboxy-terminal portion of UNC-5. The corresponding cDNAfragment, amplified by polymerase chain reaction from an embryonic humanbrain cDNA library (Stratagene), was used to screen the library,resulting in the isolation of a 3.8 kB cDNA clone comprising all but thefirst 440 nt of the coding region of the human homologue of UNC5H1.Non-overlapping probes from this cDNA were used to screen an E18 ratbrain library (gift of S. Nakanishi), leading to isolation of sevenpartial and one full length UNC5H1 cDNA and one full length UNC5H2 cDNA.Additional screens of E13 rat dorsal and ventral spinal cord librariesresulted in isolation of a second full length UNC5H2 cDNA as well as anearly full length UNC5H1 cDNA. Sequencing was performed on a Licor(L4000) automated sequencer as well as by ³³P cycle sequencing. Genbankaccession numbers are U87305 and U87306 for rUNC5H1 and rUNC5H2respectively. RNA in situ hybridization was performed as described¹³.

[0028] Antibodies, expression constructs and immunohistochemistry.Rabbit polyclonal antisera were raised to a peptide corresponding to asequence (YLRKNFEQEPLAKE, SEQ ID NO:7, residues 148-161) in theextracellular domain of UNC5H-2 that is almost completely conserved inUNC5H-1 (one amino acid substitution), and to peptides corresponding tounique sequences in the cytoplasmic domains of UNC5H-1 (GEPSPDSWSLRLKKQ,SEQ ID NO:5, residues 580-594) and UNC5H-2 (EARQQDDGDLNSLASA, SEQ IDNO:7, residues 909-924). Antisera were affinity-purified on therespective peptides (Quality Controlled Biochemicals). cDNAs for thevarious constructs were subcloned into the COS cell expression vectorpMT21 and the 293-EBNA cell expression vector pCEP4 (Invitrogen), andtransiently transfected into those cells using lipofectamine. Theantiserumto the extracellularpeptide can detect both UNC5H proteinsexpressed in transfected cells without cell permeabilization, whereasthe antisera directed against the cytoplasmic domain peptides detectedtheir respective proteins after cell permeabilization. Netrin-1 proteinwas produced, purified, used and visualized in binding assays asdescribed¹³, except that a monoclonal antibody (9E10)²⁹ directed to aC-terminal myc-epitope tag was used to detect recombinant netrin-1, andheparin was used at 1 μg/ml. A 293-EBNA cell line stably expressing theUNC5H-2-Fc fusion was derived and maintained as described^(10, 13) . Thefusion protein was purified from serum-free medium conditioned for sevendays by affinity chromatography on protein A agarose. The 293 cell lineexpressing netrin-1 was as described¹³. Binding of the UNC5H-2-Fc fusionto this line was visualized using a Cy3-conjugated secondary antibody(Jackson hnmunoresearch) directed against human Fc.

[0029] References

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EXAMPLES

[0058] 1. Protocol for high throughput vertebrate UNC-5 - netrin bindingassay.

[0059] A. Reagents:

[0060] Neutralite Avidin: 20 μg/ml in PBS.

[0061] Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at roomtemperature.

[0062] Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl₂, 1%glycerol, 0.5% NP-40, 50 mM b-mercaptoethanol, 1 mg/ml BSA, cocktail ofprotease inhibitors.

[0063]³³P vertebrate UNC-5 protein 10× stock: 10⁻⁸-10⁻⁶ M “cold”vertebrate UNC-5 supplemented with 200,000-250,000 cpm of labeledvertebrate UNC-51 (Beckman counter). Place in the 4° C. microfridgeduring screening.

[0064] Protease inhibitor cocktail (1000X): 10 mg Trypsin Inhibitor (BMB# 109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg Leupeptin (BMB # 1017128), 10 mg APMSF (BMB # 917575),and 2mM NaVo₃ (Sigma # S-6508) in 10 ml of PBS.

[0065] nerin-1: 10⁻⁷-10⁻⁵ M biotinylated netrin-1 in PBS.

[0066] B. Preparation of assay plates:

[0067] Coat with 120 μl of stock N-Avidin per well overnight at 4° C.

[0068] Wash 2 times with 200 μl PBS.

[0069] Block with 150 μl of blocking buffer.

[0070] Wash 2 times with 200 μl PBS.

[0071] C. Assay:

[0072] Add 40 μl assay buffer/well.

[0073] Add 10 μl compound or extract.

[0074] Add 10 μl ³³P-UNC-5 (20-25,000 cpm/0.1-10 pmoles/well=10⁻⁹-10⁻⁷ Mfinal conc).

[0075] Shake at 25° C. for 15 minutes.

[0076] Incubate additional 45 minutes at 25° C.

[0077] Add 40 μM biotinylated netrin-1 (0.1- 10 pmoles/40 ul in assaybuffer)

[0078] Incubate 1 hour at room temperature.

[0079] Stop the reaction by washing 4 times with 200 μM PBS.

[0080] Add 150 μM scintillation cocktail.

[0081] Count in Topcount.

[0082] D. Controls for all assays (located on each plate):

[0083] a. Non-specific binding

[0084] b. Soluble (non-biotinylated netrin-1) at 80% inhibition.

[0085] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certian changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

1 8 3014 base pairs nucleic acid double linear cDNA 1 ATGGCCGTCCGGCCCGGCCT GTGGCCAGTG CTCCTGGGCA TAGTCCTCGC CGCCTGGCTT 60 CGTGGTTCGGGTGCCCAGCA GAGTGCCACG GTGGCCAATC CAGTGCCCGG TGCCAACCCC 120 GACCTGCTGCCCCACTTCCT GGTAGAGCCT GAGGACGTGT ACATTGTCAA GAACAAGCCG 180 GTGTTGTTGGTGTGCAAGGC TGTGCCTGCC ACCCAGATCT TCTTCAAGTG CAATGGGGAA 240 TGGGTCCGCCAGGTCGATCA CGTAATTGAA CGCAGCACCG ACAGCAGCAG CGGATTGCCA 300 ACCATGGAGGTCCGTATCAA CGTATCGAGG CAGCAGGTAG AGAAAGTGTT TGGGCTGGAG 360 GAATACTGGTGCCAGTGTGT GGCATGGAGC TCCTCGGGTA CCACCAAAAG TCAGAAGGCC 420 TACATCCGGATTGCCTATTT GCGCAAGAAC TTTGAGCAGG AGCCACTGGC CAAGGAAGTG 480 TCACTGGAGCAAGGCATTGT ACTACCTTGT CGCCCCCCAG AAGGAATCCC CCCAGCTGAG 540 GTGGAGTGGCTTCGAAATGA GGACCTCGTG GACCCCTCCC TCGATCCCAA TGTGTACATC 600 ACGCGGGAGCACAGCCTAGT CGTGCGTCAG GCCCGCCTGG CCGACACGGC CAACTACACC 660 TGTGTGGCCAAGAACATCGT AGCCCGTCGC CGAAGCACCT CTGCAGCGGT CATTGTTTAT 720 GTGAACGGTGGGTGGTCGAC GTGGACTGAG TGGTCCGTCT GCAGCGCCAG CTGTGGGCGT 780 GGCTGGCAGAAACGGAGCCG GAGCTGCACC AACCCGGCAC CTCTCAACGG GGGCGCCTTC 840 TGTGAGGGGCAGAATGTCCA GAAAACAGCC TGCGCCACTC TGTGCCCAGT GGATGGGAGC 900 TGGAGTTCGTGGAGTAAGTG GTCAGCCTGT GGGCTTGACT GCACCCACTG GCGGAGCCGC 960 GAGTGCTCTGACCCAGCACC CCGCAATGGA GGTGAGGAGT GTCGGGGTGC TGACCTGGAC 1020 ACCCGCAACTGTACCAGTGA CCTCTGCCTG CACACCGCTT CTTGCCCCGA GGACGTGGCT 1080 CTCTACATCGGCCTTGTCGC TGTGGCTGTG TGCCTCTTCT TGCTGTTGCT GGCCCTTGGA 1140 CTCATTTACTGTCGCAAGAA GGAAGGGCTG GACTCCGATG TGGCCGACTC GTCCATCCTC 1200 ACCTCGGGCTTCCAGCCTGT CAGCATCAAG CCCAGCAAAG CAGACAACCC CCACCTGCTC 1260 ACCATCCAGCCAGACCTCAG CACCACCACT ACCACCTACC AGGGCAGTCT ATGTTCGAGG 1320 CAGGATGGACCCAGCCCCAA GTTCCAGCTC TCTAATGGTC ACCTGCTCAG CCCACTGGGG 1380 AGTGGCCGCCATACGTTGCA CCACAGCTCA CCCACCTCTG AGGCTGAGGA CTTCGTCTCC 1440 CGCCTCTCCACCCAAAACTA CTTTCGTTCC CTGCCCCGCG GCACCAGCAA CATGGCCTAC 1500 GGGACCTTCAACTTCCTCGG GGGCCGGCTG ATGATCCCTA ATACGGGGAT CAGCCTCCTC 1560 ATACCCCCGGATGCCATCCC CCGAGGAAAG ATCTACGAGA TCTACCTCAC ACTGCACAAG 1620 CCAGAAGACGTGAGGTTGCC CCTAGCTGGC TGTCAGACCC TGCTGAGTCC AGTCGTTAGC 1680 TGTGGGCCCCCAGGAGTCCT GCTCACCCGG CCAGTCATCC TTGCAATGGA CCACTGTGGA 1740 GAGCCCAGCCCTGACAGCTG GAGTCTGCGC CTCAAAAAGC AGTCCTGCGA GGGCAGTTGG 1800 GAGGATGTGCTGCACCTTGG TGAGGAGTCA CCTTCCCACC TCTACTACTG CCAGCTGGAG 1860 GCCGGGGCCTGCTATGTCTT CACGGAGCAG CTGGGCCGCT TTGCCCTGGT AGGAGAGGCC 1920 CTCAGCGTGGCTGCCACCAA GCGCCTCAGG CTCCTTCTGT TTGCTCCCGT GGCCTGTACG 1980 TCCCTTGAGTACAACATCCG AGTGTACTGC CTACACGACA CCCACGACGC TCTCAAGGAG 2040 GTGGTGCAGCTGGAGAAGCA GCTAGGTGGA CAGCTGATCC AGGAGCCTCG CGTCCTGCAC 2100 TTCAAAGACAGTTACCACAA CCTACGTCTC TCCATCCACG ACGTGCCCAG CTCCCTGTGG 2160 AAGAGCAAGCTACTTGTCAG CTACCAGGAG ATCCCTTTTT ACCACATCTG GAACGGCACC 2220 CAGCAGTATCTGCACTGCAC CTTCACCCTG GAGCGCATCA ACGCCAGCAC CAGCGACCTG 2280 GCCTGCAAGGTGTGGGTGTG GCAGGTGGAG GGAGATGGGC AGAGCTTCAA CATCAACTTC 2340 AACATCACTAAGGACACAAG GTTTGCTGAA TTGTTGGCTC TGGAGAGTGA AGGGGGGGTC 2400 CCAGCCCTGGTGGGCCCCAG TGCCTTCAAG ATCCCCTTCC TCATTCGGCA AAAGATCATC 2460 GCCAGTCTGGACCCACCCTG CAGCCGGGGC GCCGACTGGA GAACTCTAGC CCAGAAACTT 2520 CACCTGGACAGCCATCTTAG CTTCTTTGCC TCCAAGCCCA GCCCTACAGC CATGATCCTC 2580 AACCTATGGGAGGCACGGCA CTTCCCCAAC GGCAACCTCG GCCAGCTGGC AGCAGCTGTG 2640 GCCGGACTGGGCCAACCAGA TGCTGGCCTC TTCACGGTGT CGGAGGCCGA GTGTTGAGAC 2700 CAGCCAGGCCGGTAATGCCT ACATTCTCAC CAGCTTTGAC ACCTGCCAGG GACAGGCAAA 2760 ACCAGACAGGGGCCCTTCCC CCACACCCGG GGAGAGCTGC TTGGACAGGC CCCCTCCTGG 2820 TGAAGTTGTCCCTCGATGCT GGTCCTTCAG ACCCTGCCCA AACTCCATCC CTCCATGGCC 2880 TGCCCGGCCAGGTTGGTCTA GCCACCTGCT CTCACTCTGC CCTGGTCCCA GGGCCAGAGT 2940 AGACAGTCCTGGAGCCTGGG CTGAGCCTCG CCAGCCCATC TGTGTGTGTG TGTATATGCG 3000 TGTATGCTACCTCT 3014 1787 base pairs nucleic acid double linear cDNA 2 GCAACTGTACCAGTGACCTC TGGTACACAC TGCTTCTGGC CCTGAGGACG TGGCCCTCTA 60 TGTGGGCCTCATCGCCGTGG CCGTCTGCCT GGTCCTGCTG CTGCTTGTCC TCATCCTCGT 120 TTATTGCCGGAAGAAGGAGG GGCTGGACTC AGATGTGGCT GACTCGTCCA TTCTCACCTC 180 AGGCTTCCAGCCCGTCAGCA TCTAAGCCCA GCAAAGCAGA CAACCCCCAT CTGCTCACCA 240 TCCAGCCGGACCTCAGCACC ACCACCACCA CCTACCAGGG CAGTCTCTGT CCCCGGCAGG 300 ATGGGCCCAGCCCCAAGTTC CAGCTCACCA ATGGGCACCT GCTCAGCCCC CTGGGTGGCG 360 GCCGCCACACACTGCACCAC AGCTCTCCCA CCTCTGAGGC CGAGGAGTTC GTCTCCCGCC 420 TCTCCACCCAGAACTACTTC CGCTCCCTGC CCCGAGGCAC CAGCAACATG ACCTATGGGA 480 CCTTCAACTTCCTCGGGGGC CGGCTGATGA TCCCTAATAC AGGAATCAGC CTCCTCATCC 540 CCCCAGATGCCATACCCCGA GGGAAGATCT ATGAGATCTA CCTCACGCTG CACAAGCCGG 600 AAGACGTGAGGTTGCCCCTA GCTGGCTGTC AGACCCTGCT GAGTCCCATC GTTAGCTGTG 660 GACCCCCTGGCGTCCTGCTC ACCCGGCCAG TCATCCTGGC TATGGACCAC TGTGGGGAGC 720 CCAGCCCTGACAGCTGGAGC CTGGCCCTCA AAAAGCAGTC GTGCGAGGGA GCTGGGAGGA 780 TGTCTGCACCTGGGCGAGGA GGCGCCCTCC CACCTCTACT ACTGCCAGCT GGAGGCCAGT 840 GCCTGCTACGTCTTCACCGA GCAGCTGGGC CGCTTTGCCC TGGTGGGAGA GGCCCTCAGC 900 GTGGCTGCCGCCAAGCGCCT CAAGCTGCTT CTGTTTGCGC CGGTGGCCTG CACCTCCCTC 960 GAGTACAACATCCGGGTCTA CTGCCTGCAT GACACCCACG ATGCACTCAA GGAGGTGGTG 1020 CAGCTGGAGAAGCAGCTGGG GGGACAGCTG ATCCAGGAGC CACGGGTCCT GCACTTAAGG 1080 ACAGTTACCACAACCTGCCC TATCATCCAC GATGTGCCCA GCTCCCTGTG GAAGAGTAAG 1140 CTCCTTGTCAGCTACCAGGA GATCCCCTTT TATCACATCT GGAATGGCAC GCAGCGGTAC 1200 TTGCACTGCACCTTCACCCT GGAGCGTGTC AGCCCCAGCA CTAGTGACCT GGCCTGCAAG 1260 CTGTGGGTGTGGCAGGTGGA GGGCGACGGG CAGAGCTTCA GCATCAACTT CAACATCACC 1320 AAGGACACAAGGTTTGCTGA GCTGCTGGCT CTGGAGAGTG AAGCGGGGGT CCCAGCCCTG 1380 GTGGGCCCCAGTGCCTTCAA GATCCCCTTC CTCATTCGGC AGAAGATAAT TTCCAGCCTG 1440 GACCCACCCTGTAGGCGGGG TGCCGACTGG CGGACTCTGG CCCAGAAACT CCACCTGGAC 1500 AGCCATCTCAGCTTCTTTGC CTCCAAGCCC AGCCCCACAG CCATGATCCT CAACCTGTGG 1560 GAGGCGCGGCACTTCCCCAA CGGCAACCTC AGCCAGCTGG CTGCAGCAGT GGCTGGGACT 1620 GGCCAGCAGGACGGTGGCTT CTTTCACAGT GTTCGGAGGC TGAGTGCTGA GGCCGGCCAG 1680 GCGAACACTACAATTTTACC AGTTTTGGGA ACCCACCAAG GGACAGGCAG AAGCCGGACA 1740 AGGGCTTTTCCCAAAACCGG GGAGAGTTTT TTTGGAAAAG GCCTTTT 1787 2831 base pairs nucleicacid double linear cDNA 3 ATGAGGGCCC GGAGCGGCGG GGCCGCTGCT GTGGCGCTGCTGCTCTGCTG GGATCCGACA 60 CCGAGCTTAG CAGGCATTGA CTCTGGTGCC CAGGGACTCCCAGACTCCTT CCCATCAGCA 120 CCCGCGGAGC AGCTGCCTCA CTTCCTGCTG GAACCAGAGGATGCCTACAT CGTAAAGAAC 180 AAGCCAGTGG AATTGCACTG CCGAGCCTTC CCTGCCACACAGATCTACTT CAAGTGTAAT 240 GGCGAGTGGG TTAGCCAGAA AGGCCACGTC ACGCAGGAGAGCCTGGATGA GGCCACAGGC 300 TTGCGAATAC GAGAGGTGCA GATAGAGGTG TCGCGGCAGCAGGTGGAGGA ACTTTTTGGG 360 CTCGAGGACT ACTGGTGTCA GTGCGTGGCC TGGAGCTCTTCGGGAACCAC CAAGAGTCGC 420 CGAGCCTACA TCCGCATTGC CTACTTGCGC AAGAACTTTGACCAGGAGCC TCTGGCGAAG 480 GAGGTACCCT TGGATCATGA GGTCCTTCTG CAGTGCCGCCCACCAGAGGG AGTGCCTGTG 540 GCTGAGGTGG AATGGCTCAA GAATGAAGAT GTCATCGATCCCGCTCAGGA CACTAACTTC 600 CTGCTCACCA TTGACCACAA CCTCATCATC CGCCAGGCGCGCCTCTCAGA CACAGCCAAC 660 TACACCTGTG TGGCAAAGAA TATTGTGGCC AAGCGCCGGAGCACGACGGC CACAGTCATC 720 GTCTATGTGA ACGGAGGTTG GTCCAGCTGG GCAGAATGGTCACCCTGCTC TAACCGCTGC 780 GGCCGAGGTT GGCAGAAACG TACTAGGACC TGCACCAACCCAGCCCCACT CAATGGAGGT 840 GCCTTCTGCG AGGGACAGGC TTGCCAGAAG ACGGCTTGCACCACCGTGTG CCCAGTGGAT 900 GGAGCGTGGA CTGAGTGGAG CAAGTGGTCC GCCTGCAGCACAGAGTGTGC GCACTGGCGC 960 AGCCGCGAGT GCATGGCACC GCCGCCCCAG AACGGAGGCCGTGACTGCAG CGGGACGCTA 1020 CTTGACTCCA AGAACTGCAC CGATGGGCTG TGCGTGCTGAATCAGAGAAC TCTAAACGAC 1080 CCTAAAAGCC GCCCCCTGGA GCCGTCGGGA GACGTGGCGCTGTATGCGGG CCTCGTGGTG 1140 GCCGTCTTTG TGGTTCTGGC AGTTCTCATG GCTGTAGGAGTGATCGTGTA CCGGAGAAAC 1200 TGCCGGGACT TCGACACGGA CATCACTGAC TCCTCTGCTGCCCTCACTGG TGGTTTCCAC 1260 CCCGTCAACT TCAAGACTGC AAGGCCCAGC AACCCACAGCTCCTGCACCC ATCCGCCCCT 1320 CCGGACCTAA CGGCCAGTGC TGGCATCTAC CGCGGACCTGTGTATGCCCT GCAGGACTCT 1380 GCCGACAAGA TCCCTATGAC TAATTCACCC CTTCTGGATCCCTTGCCCAG CCTCAAGATC 1440 AAGGTCTATG ACTCCAGCAC CATCGGCTCT GGGGCTGGCCTGGCTGATGG AGCCGACCTG 1500 CTGGGTGTCT TACCACCCGG TACATACCCA GGCGATTTCTCCCGGGACAC CCACTTCCTG 1560 CACCTGCGCA GCGCCAGCCT TGGTTCCCAG CACCTCCTGGGCCTCCCTCG AGACCCCAGC 1620 AGCAGTGTCA GTGGCACCTT TGGTTGCCTG GGTGGGAGGCTGACCATTCC CGGCACAGGG 1680 GTCAGCCTGT TGGTACCAAA TGGAGCCATT CCCCAGGGCAAGTTCTATGA CTTGTATCTA 1740 CGTATCAACA AGACTGAAAG CACCCTCCCA CTTTCGGAAGGTTCCCAGAC AGTATTGAGC 1800 CCCTCGGTGA CCTGCGGGCC CACGGGCCTC CTCCTGTGCCGCCCTGTTGT CCTCACTGTG 1860 CCCCACTGTG CTGAAGTCAT TGCCGGAGAC TGGATCTTCCAGCTCAAGAC CCAGGCCCAT 1920 CAGGGCCACT GGGAGGAGGT GGTGACTTTG GATGAGGAGACTCTGAACAC CCCCTGCTAC 1980 TGCCAGCTAG AGGCTAAATC CTGCCACATC CTGTTGGACCAGCTGGGTAC CTACGTGTTC 2040 ACGGGCGAGT CCTACTCCCG CTCCGCAGTC AAGCGGCTCCAGCTAGCCAT CTTCGCCCCA 2100 GCCCTCTGCA CCTCCCTGGA GTATAGTCTC AGGGTCTACTGTCTGGAGGA CACTCCTGCA 2160 GCACTGAAGG AGGTCCTAGA GCTGGAGAGG ACTCTGGGTGGCTACTTGGT GGAGGAGCCC 2220 AAGACTTTGC TCTTTAAGGA CAGTTACCAC AACCTACGCTCTCCCTCCAT GACATCCCCC 2280 ATGCCCACTG GAGGAGCAAA CTACTGGCCA AGTACCAGGAGATTCCCTTC TACCATGTGT 2340 GGAACGGCAG CCAGAAAGCC CTGCACTGCA CTTTCACCCTGGAGAGACAT AGCCTAGCCT 2400 CCACTGAGTT CACCTGTAAG GTCTGCGTGC GGCAGGTAGAAGGGGAAGGC CAGATTTTCC 2460 AGCTGCACAC CACGCTGGCT GAGACGCCTG CTGGCTCCCTGGATGCACTC TGCTCTGCCC 2520 CTGGCAATGC TGCCACCACA CAGCTGGGAC CCTATGCCTTCAAGATACCA CTGTCCATCC 2580 GCCAGAAGAT CTGCAACAGC CTGGACGCCC CCAACTCACGGGGCAATGAC TGGCGGCTGT 2640 TGGCACAGAA GCTCTCCATG GACCGGTACC TGAACTACTTCGCCACCAAA GCTAGTCCCA 2700 CAGGCGTGAT CTTAGACCTC TGGGAAGCTC GGCAGCAGGATGATGGGGAC CTCAACAGCC 2760 TGGCCAGTGC CTTGGAGGAG ATGGGCAAGA GTGAGATGCTGGTAGCCATG ACCACTGATG 2820 GCGATTGCTG A 2831 305 base pairs nucleic aciddouble linear cDNA 4 TGGATGAGGA GACCCTGAAC ACACCCTGCT ACTGCAGCTGGAGCCCAGGG CCTGTACATC 60 CTGCTGGACC AGCTGGGCAC CTACGTTTTC ACGGGCGAGTCCTATTCCCG CTCAGCAGTC 120 AAGCGGCTCC AGCTGGCCGT TTCGCCCCCG CCCTCTGCACCTCCCTGGAG TACAGCCTCC 180 GGGTCTACTG CCTGGAGGAC ACGCCTGTAG CACTGAAGGAGGTGCTGGAG CTGGAGCGGA 240 CTCTGGGCGG ATACTTGGTG GAGGAGCCGA AACCGCTAATGTTCAAGGAC AGTTACCACA 300 ACCTT 305 898 amino acids amino acid NotRelevant Not Relevant peptide 5 Met Ala Val Arg Pro Gly Leu Trp Pro ValLeu Leu Gly Ile Val Leu 1 5 10 15 Ala Ala Trp Leu Arg Gly Ser Gly AlaGln Gln Ser Ala Thr Val Ala 20 25 30 Asn Pro Val Pro Gly Ala Asn Pro AspLeu Leu Pro His Phe Leu Val 35 40 45 Glu Pro Glu Asp Val Tyr Ile Val LysAsn Lys Pro Val Leu Leu Val 50 55 60 Cys Lys Ala Val Pro Ala Thr Gln IlePhe Phe Lys Cys Asn Gly Glu 65 70 75 80 Trp Val Arg Gln Val Asp His ValIle Glu Arg Ser Thr Asp Ser Ser 85 90 95 Ser Gly Leu Pro Thr Met Glu ValArg Ile Asn Val Ser Arg Gln Gln 100 105 110 Val Glu Lys Val Phe Gly LeuGlu Glu Tyr Trp Cys Gln Cys Val Ala 115 120 125 Trp Ser Ser Ser Gly ThrThr Lys Ser Gln Lys Ala Tyr Ile Arg Ile 130 135 140 Ala Tyr Leu Arg LysAsn Phe Glu Gln Glu Pro Leu Ala Lys Glu Val 145 150 155 160 Ser Leu GluGln Gly Ile Val Leu Pro Cys Arg Pro Pro Glu Gly Ile 165 170 175 Pro ProAla Glu Val Glu Trp Leu Arg Asn Glu Asp Leu Val Asp Pro 180 185 190 SerLeu Asp Pro Asn Val Tyr Ile Thr Arg Glu His Ser Leu Val Val 195 200 205Arg Gln Ala Arg Leu Ala Asp Thr Ala Asn Tyr Thr Cys Val Ala Lys 210 215220 Asn Ile Val Ala Arg Arg Arg Ser Thr Ser Ala Ala Val Ile Val Tyr 225230 235 240 Val Asn Gly Gly Trp Ser Thr Trp Thr Glu Trp Ser Val Cys SerAla 245 250 255 Ser Cys Gly Arg Gly Trp Gln Lys Arg Ser Arg Ser Cys ThrAsn Pro 260 265 270 Ala Pro Leu Asn Gly Gly Ala Phe Cys Glu Gly Gln AsnVal Gln Lys 275 280 285 Thr Ala Cys Ala Thr Leu Cys Pro Val Asp Gly SerTrp Ser Ser Trp 290 295 300 Ser Lys Trp Ser Ala Cys Gly Leu Asp Cys ThrHis Trp Arg Ser Arg 305 310 315 320 Glu Cys Ser Asp Pro Ala Pro Arg AsnGly Gly Glu Glu Cys Arg Gly 325 330 335 Ala Asp Leu Asp Thr Arg Asn CysThr Ser Asp Leu Cys Leu His Thr 340 345 350 Ala Ser Cys Pro Glu Asp ValAla Leu Tyr Ile Gly Leu Val Ala Val 355 360 365 Ala Val Cys Leu Phe LeuLeu Leu Leu Ala Leu Gly Leu Ile Tyr Cys 370 375 380 Arg Lys Lys Glu GlyLeu Asp Ser Asp Val Ala Asp Ser Ser Ile Leu 385 390 395 400 Thr Ser GlyPhe Gln Pro Val Ser Ile Lys Pro Ser Lys Ala Asp Asn 405 410 415 Pro HisLeu Leu Thr Ile Gln Pro Asp Leu Ser Thr Thr Thr Thr Thr 420 425 430 TyrGln Gly Ser Leu Cys Ser Arg Gln Asp Gly Pro Ser Pro Lys Phe 435 440 445Gln Leu Ser Asn Gly His Leu Leu Ser Pro Leu Gly Ser Gly Arg His 450 455460 Thr Leu His His Ser Ser Pro Thr Ser Glu Ala Glu Asp Phe Val Ser 465470 475 480 Arg Leu Ser Thr Gln Asn Tyr Phe Arg Ser Leu Pro Arg Gly ThrSer 485 490 495 Asn Met Ala Tyr Gly Thr Phe Asn Phe Leu Gly Gly Arg LeuMet Ile 500 505 510 Pro Asn Thr Gly Ile Ser Leu Leu Ile Pro Pro Asp AlaIle Pro Arg 515 520 525 Gly Lys Ile Tyr Glu Ile Tyr Leu Thr Leu His LysPro Glu Asp Val 530 535 540 Arg Leu Pro Leu Ala Gly Cys Gln Thr Leu LeuSer Pro Val Val Ser 545 550 555 560 Cys Gly Pro Pro Gly Val Leu Leu ThrArg Pro Val Ile Leu Ala Met 565 570 575 Asp His Cys Gly Glu Pro Ser ProAsp Ser Trp Ser Leu Arg Leu Lys 580 585 590 Lys Gln Ser Cys Glu Gly SerTrp Glu Asp Val Leu His Leu Gly Glu 595 600 605 Glu Ser Pro Ser His LeuTyr Tyr Cys Gln Leu Glu Ala Gly Ala Cys 610 615 620 Tyr Val Phe Thr GluGln Leu Gly Arg Phe Ala Leu Val Gly Glu Ala 625 630 635 640 Leu Ser ValAla Ala Thr Lys Arg Leu Arg Leu Leu Leu Phe Ala Pro 645 650 655 Val AlaCys Thr Ser Leu Glu Tyr Asn Ile Arg Val Tyr Cys Leu His 660 665 670 AspThr His Asp Ala Leu Lys Glu Val Val Gln Leu Glu Lys Gln Leu 675 680 685Gly Gly Gln Leu Ile Gln Glu Pro Arg Val Leu His Phe Lys Asp Ser 690 695700 Tyr His Asn Leu Arg Leu Ser Ile His Asp Val Pro Ser Ser Leu Trp 705710 715 720 Lys Ser Lys Leu Leu Val Ser Tyr Gln Glu Ile Pro Phe Tyr HisIle 725 730 735 Trp Asn Gly Thr Gln Gln Tyr Leu His Cys Thr Phe Thr LeuGlu Arg 740 745 750 Ile Asn Ala Ser Thr Ser Asp Leu Ala Cys Lys Val TrpVal Trp Gln 755 760 765 Val Glu Gly Asp Gly Gln Ser Phe Asn Ile Asn PheAsn Ile Thr Lys 770 775 780 Asp Thr Arg Phe Ala Glu Leu Leu Ala Leu GluSer Glu Gly Gly Val 785 790 795 800 Pro Ala Leu Val Gly Pro Ser Ala PheLys Ile Pro Phe Leu Ile Arg 805 810 815 Gln Lys Ile Ile Ala Ser Leu AspPro Pro Cys Ser Arg Gly Ala Asp 820 825 830 Trp Arg Thr Leu Ala Gln LysLeu His Leu Asp Ser His Leu Ser Phe 835 840 845 Phe Ala Ser Lys Pro SerPro Thr Ala Met Ile Leu Asn Leu Trp Glu 850 855 860 Ala Arg His Phe ProAsn Gly Asn Leu Gly Gln Leu Ala Ala Ala Val 865 870 875 880 Ala Gly LeuGly Gln Pro Asp Ala Gly Leu Phe Thr Val Ser Glu Ala 885 890 895 Glu Cys557 amino acids amino acid Not Relevant Not Relevant peptide 6 Asn CysThr Ser Asp Leu Xaa Val His Thr Ala Ser Gly Pro Glu Asp 1 5 10 15 ValAla Leu Tyr Val Gly Leu Ile Ala Val Ala Val Cys Leu Val Leu 20 25 30 LeuLeu Leu Val Leu Ile Leu Val Tyr Cys Arg Lys Lys Glu Gly Leu 35 40 45 AspSer Asp Val Ala Asp Ser Ser Ile Leu Thr Ser Gly Phe Gln Pro 50 55 60 ValSer Ile Lys Pro Ser Lys Ala Asp Asn Pro His Leu Leu Thr Ile 65 70 75 80Gln Pro Asp Leu Ser Thr Thr Thr Thr Thr Tyr Gln Gly Ser Leu Cys 85 90 95Pro Arg Gln Asp Gly Pro Ser Pro Lys Phe Gln Leu Thr Asn Gly His 100 105110 Leu Leu Ser Pro Leu Gly Gly Gly Arg His Thr Leu His His Ser Ser 115120 125 Pro Thr Ser Glu Ala Glu Glu Phe Val Ser Arg Leu Ser Thr Gln Asn130 135 140 Tyr Phe Arg Ser Leu Pro Arg Gly Thr Ser Asn Met Thr Tyr GlyThr 145 150 155 160 Phe Asn Phe Leu Gly Gly Arg Leu Met Ile Pro Asn ThrGly Ile Ser 165 170 175 Leu Leu Ile Pro Pro Asp Ala Ile Pro Arg Gly LysIle Tyr Glu Ile 180 185 190 Tyr Leu Thr Leu His Lys Pro Glu Asp Val ArgLeu Pro Leu Ala Gly 195 200 205 Cys Gln Thr Leu Leu Ser Pro Ile Val SerCys Gly Pro Pro Gly Val 210 215 220 Leu Leu Thr Arg Pro Val Ile Leu AlaMet Asp His Cys Gly Glu Pro 225 230 235 240 Ser Pro Asp Ser Trp Ser LeuAla Leu Lys Lys Gln Ser Cys Glu Gly 245 250 255 Ser Trp Glu Asp Val LeuHis Leu Gly Glu Glu Ala Pro Ser His Leu 260 265 270 Tyr Tyr Cys Gln LeuGlu Ala Ser Ala Cys Tyr Val Phe Thr Glu Gln 275 280 285 Leu Gly Arg PheAla Leu Val Gly Glu Ala Leu Ser Val Ala Ala Ala 290 295 300 Lys Arg LeuLys Leu Leu Leu Phe Ala Pro Val Ala Cys Thr Ser Leu 305 310 315 320 GluTyr Asn Ile Arg Val Tyr Cys Leu His Asp Thr His Asp Ala Leu 325 330 335Lys Glu Val Val Gln Leu Glu Lys Gln Leu Gly Gly Gln Leu Ile Gln 340 345350 Glu Pro Arg Val Leu His Leu Xaa Asp Ser Tyr His Asn Leu Xaa Leu 355360 365 Ser Xaa His Asp Val Pro Ser Ser Leu Trp Lys Ser Lys Leu Leu Val370 375 380 Ser Tyr Gln Glu Ile Pro Phe Tyr His Ile Trp Asn Gly Thr GlnArg 385 390 395 400 Tyr Leu His Cys Thr Phe Thr Leu Glu Arg Val Ser ProSer Thr Ser 405 410 415 Asp Leu Ala Cys Lys Leu Trp Val Trp Gln Val GluGly Asp Gly Gln 420 425 430 Ser Phe Ser Ile Asn Phe Asn Ile Thr Lys AspThr Arg Phe Ala Glu 435 440 445 Leu Leu Ala Leu Glu Ser Glu Ala Gly ValPro Ala Leu Val Gly Pro 450 455 460 Ser Ala Phe Lys Ile Pro Phe Leu IleArg Gln Lys Ile Ile Ser Ser 465 470 475 480 Leu Asp Pro Pro Cys Arg ArgGly Ala Asp Trp Arg Thr Leu Ala Gln 485 490 495 Lys Leu His Leu Asp SerHis Leu Ser Phe Phe Ala Ser Lys Pro Ser 500 505 510 Pro Thr Ala Met IleLeu Asn Leu Trp Glu Ala Arg His Phe Pro Asn 515 520 525 Gly Asn Leu SerGln Leu Ala Ala Ala Val Ala Gly Thr Xaa Pro Ala 530 535 540 Gly Arg TrpLeu Leu Ser Gln Cys Ser Glu Ala Glu Cys 545 550 555 943 amino acidsamino acid Not Relevant Not Relevant peptide 7 Met Arg Ala Arg Ser GlyGly Ala Ala Ala Val Ala Leu Leu Leu Cys 1 5 10 15 Trp Asp Pro Thr ProSer Leu Ala Gly Ile Asp Ser Gly Ala Gln Gly 20 25 30 Leu Pro Asp Ser PhePro Ser Ala Pro Ala Glu Gln Leu Pro His Phe 35 40 45 Leu Leu Glu Pro GluAsp Ala Tyr Ile Val Lys Asn Lys Pro Val Glu 50 55 60 Leu His Cys Arg AlaPhe Pro Ala Thr Gln Ile Tyr Phe Lys Cys Asn 65 70 75 80 Gly Glu Trp ValSer Gln Lys Gly His Val Thr Gln Glu Ser Leu Asp 85 90 95 Glu Ala Thr GlyLeu Arg Ile Arg Glu Val Gln Ile Glu Val Ser Arg 100 105 110 Gln Gln ValGlu Glu Leu Phe Gly Leu Glu Asp Tyr Trp Cys Gln Cys 115 120 125 Val AlaTrp Ser Ser Ser Gly Thr Thr Lys Ser Arg Arg Ala Tyr Ile 130 135 140 ArgIle Ala Tyr Leu Arg Lys Asn Phe Asp Gln Glu Pro Leu Ala Lys 145 150 155160 Glu Val Pro Leu Asp His Glu Val Leu Leu Gln Cys Arg Pro Pro Glu 165170 175 Gly Val Pro Val Ala Glu Val Glu Trp Leu Lys Asn Glu Asp Val Ile180 185 190 Asp Pro Ala Gln Asp Thr Asn Phe Leu Leu Thr Ile Asp His AsnLeu 195 200 205 Ile Ile Arg Gln Ala Arg Leu Ser Asp Thr Ala Asn Tyr ThrCys Val 210 215 220 Ala Lys Asn Ile Val Ala Lys Arg Arg Ser Thr Thr AlaThr Val Ile 225 230 235 240 Val Tyr Val Asn Gly Gly Trp Ser Ser Trp AlaGlu Trp Ser Pro Cys 245 250 255 Ser Asn Arg Cys Gly Arg Gly Trp Gln LysArg Thr Arg Thr Cys Thr 260 265 270 Asn Pro Ala Pro Leu Asn Gly Gly AlaPhe Cys Glu Gly Gln Ala Cys 275 280 285 Gln Lys Thr Ala Cys Thr Thr ValCys Pro Val Asp Gly Ala Trp Thr 290 295 300 Glu Trp Ser Lys Trp Ser AlaCys Ser Thr Glu Cys Ala His Trp Arg 305 310 315 320 Ser Arg Glu Cys MetAla Pro Pro Pro Gln Asn Gly Gly Arg Asp Cys 325 330 335 Ser Gly Thr LeuLeu Asp Ser Lys Asn Cys Thr Asp Gly Leu Cys Val 340 345 350 Leu Asn GlnArg Thr Leu Asn Asp Pro Lys Ser Arg Pro Leu Glu Pro 355 360 365 Ser GlyAsp Val Ala Leu Tyr Ala Gly Leu Val Val Ala Val Phe Val 370 375 380 ValLeu Ala Val Leu Met Ala Val Gly Val Ile Val Tyr Arg Arg Asn 385 390 395400 Cys Arg Asp Phe Asp Thr Asp Ile Thr Asp Ser Ser Ala Ala Leu Thr 405410 415 Gly Gly Phe His Pro Val Asn Phe Lys Thr Ala Arg Pro Ser Asn Pro420 425 430 Gln Leu Leu His Pro Ser Ala Pro Pro Asp Leu Thr Ala Ser AlaGly 435 440 445 Ile Tyr Arg Gly Pro Val Tyr Ala Leu Gln Asp Ser Ala AspLys Ile 450 455 460 Pro Met Thr Asn Ser Pro Leu Leu Asp Pro Leu Pro SerLeu Lys Ile 465 470 475 480 Lys Val Tyr Asp Ser Ser Thr Ile Gly Ser GlyAla Gly Leu Ala Asp 485 490 495 Gly Ala Asp Leu Leu Gly Val Leu Pro ProGly Thr Tyr Pro Gly Asp 500 505 510 Phe Ser Arg Asp Thr His Phe Leu HisLeu Arg Ser Ala Ser Leu Gly 515 520 525 Ser Gln His Leu Leu Gly Leu ProArg Asp Pro Ser Ser Ser Val Ser 530 535 540 Gly Thr Phe Gly Cys Leu GlyGly Arg Leu Thr Ile Pro Gly Thr Gly 545 550 555 560 Val Ser Leu Leu ValPro Asn Gly Ala Ile Pro Gln Gly Lys Phe Tyr 565 570 575 Asp Leu Tyr LeuArg Ile Asn Lys Thr Glu Ser Thr Leu Pro Leu Ser 580 585 590 Glu Gly SerGln Thr Val Leu Ser Pro Ser Val Thr Cys Gly Pro Thr 595 600 605 Gly LeuLeu Leu Cys Arg Pro Val Val Leu Thr Val Pro His Cys Ala 610 615 620 GluVal Ile Ala Gly Asp Trp Ile Phe Gln Leu Lys Thr Gln Ala His 625 630 635640 Gln Gly His Trp Glu Glu Val Val Thr Leu Asp Glu Glu Thr Leu Asn 645650 655 Thr Pro Cys Tyr Cys Gln Leu Glu Ala Lys Ser Cys His Ile Leu Leu660 665 670 Asp Gln Leu Gly Thr Tyr Val Phe Thr Gly Glu Ser Tyr Ser ArgSer 675 680 685 Ala Val Lys Arg Leu Gln Leu Ala Ile Phe Ala Pro Ala LeuCys Thr 690 695 700 Ser Leu Glu Tyr Ser Leu Arg Val Tyr Cys Leu Glu AspThr Pro Ala 705 710 715 720 Ala Leu Lys Glu Val Leu Glu Leu Glu Arg ThrLeu Gly Gly Tyr Leu 725 730 735 Val Glu Glu Pro Lys Thr Leu Leu Phe LysAsp Ser Tyr His Asn Leu 740 745 750 Arg Leu Ser Leu His Asp Ile Pro HisAla His Trp Arg Ser Lys Leu 755 760 765 Leu Ala Lys Tyr Gln Glu Ile ProPhe Tyr His Val Trp Asn Gly Ser 770 775 780 Gln Lys Ala Leu His Cys ThrPhe Thr Leu Glu Arg His Ser Leu Ala 785 790 795 800 Ser Thr Glu Phe ThrCys Lys Val Cys Val Arg Gln Val Glu Gly Glu 805 810 815 Gly Gln Ile PheGln Leu His Thr Thr Leu Ala Glu Thr Pro Ala Gly 820 825 830 Ser Leu AspAla Leu Cys Ser Ala Pro Gly Asn Ala Ala Thr Thr Gln 835 840 845 Leu GlyPro Tyr Ala Phe Lys Ile Pro Leu Ser Ile Arg Gln Lys Ile 850 855 860 CysAsn Ser Leu Asp Ala Pro Asn Ser Arg Gly Asn Asp Trp Arg Leu 865 870 875880 Leu Ala Gln Lys Leu Ser Met Asp Arg Tyr Leu Asn Tyr Phe Ala Thr 885890 895 Lys Ala Ser Pro Thr Gly Val Ile Leu Asp Leu Trp Glu Ala Arg Gln900 905 910 Gln Asp Asp Gly Asp Leu Asn Ser Leu Ala Ser Ala Leu Glu GluMet 915 920 925 Gly Lys Ser Glu Met Leu Val Ala Met Thr Thr Asp Gly AspCys 930 935 940 102 amino acids amino acid Not Relevant Not Relevantpeptide 8 Asp Glu Glu Thr Leu Asn Thr Pro Cys Tyr Xaa Gln Leu Glu ProArg 1 5 10 15 Ala Cys Xaa Ile Leu Leu Asp Gln Leu Gly Thr Tyr Val PheThr Gly 20 25 30 Glu Ser Tyr Ser Arg Ser Ala Val Lys Arg Leu Gln Leu AlaVal Phe 35 40 45 Ala Pro Ala Leu Cys Thr Ser Leu Glu Tyr Ser Leu Arg ValTyr Cys 50 55 60 Leu Glu Asp Thr Pro Val Ala Leu Lys Glu Val Leu Glu LeuGlu Arg 65 70 75 80 Thr Leu Gly Gly Tyr Leu Val Glu Glu Pro Lys Pro LeuMet Phe Lys 85 90 95 Asp Ser Tyr His Asn Leu 100

What is claimed is:
 1. An isolated vertebrate UNC-5 protein comprisingSEQ ID NO:5, 6, 7 or, 8, or a fragment thereof having vertebrateUNC-5-specific activity.
 2. An isolated protein according to claim 1,wherein said protein specifically binds a natural netrin protein.
 3. Arecombinant nucleic acid encoding a protein according to claim
 1. 4. Acell comprising a nucleic acid according to claim
 3. 5. A method ofmaking an isolated vertebrate UNC-5 protein, comprising steps:introducing a nucleic acid according to claim 3 into a host cell orcellular extract, incubating said host cell or extract under conditionswhereby said nucleic acid is expressed as a transcript and saidtranscript is expressed as a translation product comprising saidprotein, and isolating said translation product.
 6. An isolatedvertebrate UNC-5 protein made by the method of claim
 5. 7. An isolatedvertebrate unc-5 nucleic acid comprising SEQ ID NO:1 ,2, 3, or 4, or afragment thereof having at least 24 consecutive bases of SEQ ID NO:1, 2,3, or 4 and sufficient to specifically hybridize with a nucleic acidhaving the sequence of the corresponding SEQ ID NO:1, 2, 3, or 4 in thepresence of natural C. elegans unc-5 cDNA.
 8. A method of screening foran agent which modulates the binding of a vertebrate UNC-5 protein to abinding target, said method comprising the steps of: incubating amixture comprising: an isolated protein according to claim 1, a bindingtarget of said protein, and a candidate agent; under conditions whereby,but for the presence of said agent, said protein specifically binds saidbinding target at a reference affinity; detecting the binding affinityof said protein to said binding target to determine an agent-biasedaffinity, wherein a difference between the agent-biased affinity and thereference affinity indicates that said agent modulates the binding ofsaid protein to said binding target.
 9. A method according to claim 8,wherein said binding target is a natural netrin protein.